DOI 10.1007/s00572-003-0254-5
O R I G I N A L P A P E R
Cristina Cruz · James J. Green · Christine A. Watson ·
Frederick Wilson · Maria Amlia Martins-Lou¼o
Functional aspects of root architecture and mycorrhizal inoculation
with respect to nutrient uptake capacity
Received: 27 January 2003 / Accepted: 27 May 2003 / Published online: 10 July 2003 Springer-Verlag 2003
Abstract
The aim of this research was to investigate the
effect of arbuscular mycorrhizal (AM) colonisation on
root morphology and nitrogen uptake capacity of carob
(Ceratonia siliqua L.) under high and low nutrient
conditions. The experimental design was a factorial
arrangement of presence/absence of mycorrhizal fungus
inoculation (Glomus intraradices) and high/low nutrient
status. Percent AM colonisation, nitrate and ammonium
uptake capacity, and nitrogen and phosphorus contents
were determined in 3-month-old seedlings. Grayscale and
colour images were used to study root morphology and
topology, and to assess the relation between root
pigmentation and physiological activities. AM
colonisa-tion lead to a higher allocacolonisa-tion of biomass to white and
yellow parts of the root. Inorganic nitrogen uptake
capacity per unit root length and nitrogen content were
greatest in AM colonised plants grown under low nutrient
conditions. A better match was found between plant
nitrogen content and biomass accumulation, than between
plant phosphorus content and biomass accumulation. It is
suggested that the increase in nutrient uptake capacity of
AM colonised roots is dependent both on changes in root
morphology and physiological uptake potential. This
study contributes to an understanding of the role of AM
fungi and root morphology in plant nutrient uptake and
shows that AM colonisation improves the nitrogen
nutrition of plants, mainly when growing at low levels
of nutrients.
Keywords
Arbuscular mycorrhizae · Carob · Ceratonia
siliqua L. · Nitrogen uptake · Root architecture
Introduction
Most research on the importance of arbuscular
mycor-rhizal (AM) associations to mineral plant nutrition has
focused on their ability to improve plant growth by
enhancing phosphorus uptake (Fitter and Hay 2002).
Several works have shown that AM fungi can interfere
with nitrogen uptake and metabolism (Smith and Read
1997). Nevertheless, there is still controversy concerning
their contribution to the nutritional status of the plant
(Bouma et al. 2001).
Root architecture, morphology and physiology are key
factors in plant productivity and can provide useful
information for an appreciation of root nutrient absorbing
capacity, especially in environments with low nutrient
availability (Lynch 1995). Root system development can
be analysed using various mathematical or developmental
models (Tisserant et al. 1992). However, these models do
not relate functional aspects to root architecture and
morphology. To generate hypotheses on root function and
on root strategies, topological analyses combined with
physiological studies are required.
Many environmental factors such as soil moisture and
nutrient concentration have been considered to influence
the topological pattern of root systems (Fitter 1986). AM
associations have been shown to induce modifications in
root architecture and morphogenesis in herbaceous plants
as well as in trees (Berta et al. 1990; Schellenbaum et al.
1991; Tisserant et al. 1992), but reports are inconsistent
on the kind of modifications imposed by AM association
(Barker et al. 1998; Smilauerova and Smilauer 2002).
However, very little is known about the way mycorrhiza
influence root capacity for nitrogen uptake.
This research follows a series of studies of changes in
the morphology and physiology of carob (Ceratonia
siliqua L.) roots (Cruz et al. 1995, 1997). Carob is a
slow-growing evergreen, sclerophylous, Mediterranean woody
C. Cruz (
)
) · M. A. Martins-Lou¼oCentro de Ecologia e Biologia Vegetal (CEBV), Departamento de Biologia Vegetal,
Faculdade de CiÞncias da Universidade de Lisboa,
Campo Grande, Bloco C-2 Piso 4, 1749–016 Lisboa, Portugal e-mail: [email protected]
Tel.: +351-21-7500000 Fax: +351-21-7500048
J. J. Green · C. A. Watson · F. Wilson
Scottish Agricultural College, Craibstone Estate, Aberdeen, AB21 9TQ, UK
plant (Martins-Lou¼o and Brito de Carvalho 1990) well
adapted to the marginal nutrient-poor areas of
Mediter-ranean ecosystems.
The aim of this work was to compare the capacity of
non-AM roots and AM roots to take up nitrogen, and to
assess the effects of AM colonisation on root morphology
and architecture, relating measurements of root
architec-ture with root function.
Materials and Methods
The inoculum
The inoculum was an isolate of Glomus intraradices (Smith and Schenck) which has been in the collection of the del Zaidin Experimental Station, Granada, Spain for over 25 years. Prior to the experiment the inoculum was multiplied by pot culture with maize (Brundrett 1996). The growth medium for the pot cultures was a 1:9 mix of sieved soil (2 mm) and sand, sterilised by autoclaving. The soil was a silty clay loam topsoil (Panholes series, Rothamsted Estate, Hertfordshire, UK) selected as a surrogate for Mediter-ranean soil for its high similarity with the MediterMediter-ranean soils: high pH (7.8 in 0.01 M CaCl2), fine texture and low organic matter
content. A non-AM inoculum was produced by growing maize in the same soil mixture after sterilisation and without propagules. Maize plants were allowed to grow for 6 weeks, after which the shoot was harvested and discarded. The roots were cut into small segments and mixed with the soil, ensuring the same proportion of root material to soil dry weight in mycorrhizal and non-mycorrhizal treatments. The soil with the maize roots was used to grow the carob plants. The nitrogen, phosphorus, potassium and magnesium concentrations of the substrate were 33, 200, 310 and 80 mg kg-1
substrate, respectively.
Plant material, growth conditions and experimental design Seeds of carob ( Ceratonia siliqua L. cv. Mulata), harvested in 1999, were obtained from the Associa¼o Interprofissional para o Desenvolvimento da Produ¼o e Valoriza¼o da Alfarroba (Al-garve, Portugal). Seed dormancy was broken by placing the seeds in concentrated sulphuric acid (97%) for 15 min. The seeds were then washed and imbibed in tap water for 48 h, germinated in an incubator at 25C and transplanted to treatment containers once the radicle had emerged. Treatment containers were cylindrical, 11 cm in diameter and 50 cm deep (volume 4.5 l), made from two lengths of plastic guttering joined with silicon sealant. These could be opened, allowing the root systems to be extracted intact on harvesting .
During the 3 months of the experiment, plants were grown in a glasshouse under a photoperiod of 16 h, with photosynthetic active radiation of 350 mmol m-2 s-1 at seedling level, mean daily
maximum temperature 24C and mean minimum temperature 18C. Containers were initially watered to field capacity at the start of the study, and watered with 50 ml water every 3 days. Watering solutions were either deionised water (low nutrients) or Hoagland’s solution (high nutrients, solution no.1; Hewitt 1966) which comprises a balanced nutrient solution (nitrogen as 0.611 g NO3
-l-1, phosphorus as 0.031 g PO
43- l-1 and 0.236 g K+ l-1) with an
extensive micronutrient supplement (Hewitt 1966). In total, each plant (one per pot) received 1.5 l deionised water or nutrient solution, and plants from high nutrient-level treatments received a supplement of 300, 900, 480 and 340 mg plant-1of phosphorus,
nitrogen, potassium and magnesium, respectively.
The experimental design was a factorial arrangement of mycorrhiza inoculation and nutrition. The four treatments were: (1) no inoculation and no additional nutrition (NML); (2) no inoculation and additional nutrition (NMH); (3) mycorrhiza
inoc-ulum and no additional nutrition (ML); (4) mycorrhiza inocinoc-ulum and additional nutrition (MH). There were ten replicates of each treatment but due to root breakage during extraction only four replicates were used for the root morphology analyses. The roots used for morphology analyses were later used for the determination of nutrient concentrations. The remaining six replicates were used for determination of AM colonisation and nutrient uptake.
Growth morphology
Three-month-old plants were harvested and total fresh weight determined. Plants were then divided into root and shoot and each part weighed again. Samples of five plants per treatment were oven dried at 50C, until constant weight, and then weighed again. The root to shoot ratios were then calculated on a dry weight basis. The dry material was used to determine plant nitrogen and phosphorus contents. Nitrogen was determined by carbon/nitrogen elemental analyses, and phosphorus by the molybdophosphate method (Wantanabe and Olsen 1965).
AM colonisation
For each root sub-sample, three different groups of root segments were prepared according to their colour and root zones : white (apical region), yellow (elongation zones) and brown (nature zone) (Cruz et al. 1995). After collection, roots were stored in 50% ethanol. Mycorrhiza staining (Correia and Martins-Lou¼o 1997) was initiated by heating the roots in 10% KOH for 60 min at 90C and washing them in water. Roots were bleached in alkaline H2O2
for 2 days, after which they were rinsed in water and soaked in 10% HCl for 10 min. They were then stained in acidic glycerol containing 0.05% fuschin for 15 min at 75C. Acidic glycerol was used for destaining. The degree of mycorrhizal infection in roots was assessed by spreading the root sample evenly in a Petri dish, where a grid (0.4 cm side squares) had been previously marked. The Petri dish was observed under the dissecting microscope at a low magnification. First the number of intersects of the root with the grid, and then the intersects of infected roots with the grid were counted. The percentage of infection in the root was calculated by the ratio between the number of intersects with infection and the total number of intersects. Each Petri dish was observed 5 times, and the average taken as the degree of root infection.
Nitrate and ammonium uptake
Nitrogen uptake through defined root regions was studied with roots of carob seedlings grown as described above. All the experiments were performed between 1000 and 1400 hours in order to avoid errors due to diurnal variations. The roots were rinsed in water and root segments of the same colour (white, yellow and brown) obtained from five different plants were cut into pieces of 1 cm and mixed. Samples of segments (0.3–1 g) were weighed and transferred to Petri dishes containing 10 ml of 25% modified Crone solution (Lazof et al. 1992; Cruz et al. 1995; Gessler et al. 1998). The media were gently aerated to maintain adequate oxygen supply and minimise boundary layers around the root segments. Three Petri dishes with root medium but without root segments were used to calculate the loss of nitrate and ammonium not due to absorption by roots. Twenty Petri dishes were prepared for each group of root sections. Every 30 min, during 2 h, five Petri dishes were collected, and solution volumes and nitrogen concentrations of the medium were determined. The values reported are the means of the results obtained.
Determinations of net uptake rates of nitrate and ammonium were based on nitrogen disappearance from the solution per unit time. The rates were expressed as mmol g-1root fresh weight h-1and
calculated from the changes in nitrogen concentration and solution volumes at consecutive sampling times.
Nitrate was determined after reduction to nitrite (McNamara et al. 1971), nitrite according to Snell and Snell (1949) and ammonium by the indophenol-blue reaction (Solozano 1969).
Root morphology
Cleaned root systems were arranged to minimise overlaps in an A3-sized perspex tray and scanned in both grayscale [transparency adapter, 150 dpi, tagged image file (TIF) format] and colour (reflective, blue background, 96 dpi, TIF format). Where necessary, root systems were physically dissected into up to four parts and the images rejoined, before analysis with image analysis software (WinRhizo; Regent Instruments, Quebec). Grayscale images were used to measure total length, link length and topology of the root systems. The root was considered, according to Fitter et al. (1988), as a set of links. Links or internodes were defined as linear portions between a terminal meristem and a branching point (external link) or between two branching points (internal link). Two types of internal links were considered, internal links adjacent to external links were referred to as external-internal; and those without connection to a link with a meristem were referred to as internal-internal links. Root topology was characterised by the magnitude (number of external links) and by altitude ( a; the number of links in the longest path between an external link and the base link). The topological indices used, calculated according to Werner and Smart (1973), were the ratio of a to the expected a [E( a)], i.e. [ a/E( a)] and the ratio of pathlength (Pe) to the expected Pe [E(Pe)], i.e. [Pe/ E(Pe)] (Fitter et al. 1988). At magnitudes >200 we used a linear extrapolation of the Werner and Smart data {E( a)=0.299+0.593log magnitude, r 2=0.999, P <0.001, and log [E(Pe)]=0.223+1.51log magnitude, r2=1.000, P <0.001} to calculate expected values, but the relationship is not thought to stray from the linear. The topological indices indicate the degree of branching on a spectrum between the theoretical extremes of dichotomously branched and herringbone structures. Increases in a/E( a) and Pe/E(Pe) indicate more herringbone roots.
The colour images were used to measure the length of white tip and yellow-brown elongation zone in the root systems (hereafter referred to as the lightly pigmented zone). The brown and dark brown parts of the roots were designated as heavily pigmented according to our previous knowledge of carob root morphology (M. A. Martins-Lou¼o, unpublished data). The two root categories were distinguished from the background and the rest of the root system using a colour group selected from a scanned soil colour chart (Munsell 1975): white tips 10YR 7/1–8/4 and elongation zone 10 YR 3/3–8/7.
Data were analysed by two-way ANOVA. Link lengths and topological indices were analysed by analysis of covariance with log(number of tips) as the covariate, because the indices are scale dependant (Fitter et al. 1988; Green et al. 2003).
Calculations
Calculation of the potential nitrogen uptake rate of each root section was based on the product of the nitrogen uptake rate per unit weight of the root section and the weight of the root section per root system.
The root to shoot ratio was calculated as the ratio between root dry matter and shoot dry matter.
Results
Biomass production by carob plants inoculated with AM
fungi was greater under low levels of nutrients, i.e. about
30% dry weight (Fig. 1). Among the other treatments no
significant differences were observed with or without
mycorrhiza.
Uninoculated plants developed <1% root AM
coloni-sation, higher percentages were observed for the plants
exposed to the AM inoculum (Table 1). AM colonisation
was higher (30–36% compared with 11–12%) for the
inoculated plants grown with low than those grown with
Fig. 1 Plant biomass, root biomass and root to shoot ratio (R/S) of 3-month-old carob seedlings [non-mycorrhizal (grey bars) or mycorrhizal (black bars)] grown with low or high nutrient supply to the root medium. Within panels, values with the same letter are not significantly different according to Fisher’s LSD test at P=0.05. DW Dry weight
Table 1 Percentage of root infected with arbuscular mycorrhizal (AM) fungi according to treatments: no inoculation and no additional nutrition (NML); no inoculation and additional nutrition (NMH); mycorrhiza inoculum and no additional nutrition (ML); mycorrhiza inoculum and additional nutrition (MH). Root systems of Ceratonia siliqua grown with AM inoculation and nutrient treatments were divided into three zones according to their colour. Values with the same letter are not significantly different according to Fisher’s LSD test at P=0.05. Probabilities from the ANOVA are given (df=1, 20)
Treatment Root zone (colour)
White Yellow Brown
NML 0.9 c 0.9 c 0.4 c NMH 0.7 c 0.9 c 0.3 c ML 30.0 a 36.0 a 5.6 a MH 12.7 b 11.9 b 2.8 b Source P P P AM 0.010 0.059 0.001 Nutrients 0.005 0.000 0.001 AMNutrients 0.011 0.058 0.001
high levels of nutrients (Table 1). Most of the AM
colonisation was observed in the lightly pigmented (white
and yellow) parts of the root.
Dry weight root to shoot ratios were significantly
higher for non-mycorrhizal plants at a low nutrient level
(Fig. 1). No significant differences in root to shoot ratio
were observed among plants from the other treatments
(NMH, ML or MH).
Nevertheless, roots of the four treatments were quite
different. Roots of the treatments without the nutrient
supplement (NML and ML) appeared to be more brittle
than those of the treatments receiving more nutrients
(NMH and MH). This was partly due to a higher
allocation of biomass to lightly pigmented root regions
(Table 2). In NML and ML treatments, the weight of the
lightly pigmented root regions (white and yellow parts of
the root) represented 60% and 68% of the total fresh
weight of the root, while for the NMH and MH treatments
the same root fraction corresponded to 48% and 59%,
respectively. The greater proportion of lightly pigmented
root regions (white tips and yellow zone) in root systems
under low nutrient levels was confirmed on a root length
basis (Table 3).
Nitrate and ammonium uptake capacities of excised
roots, on a fresh weight basis, were higher for the white
part of the roots, in contrast to the very limited uptake by
the brown parts of the root (Fig. 2). The effects of
increased levels of nutrients and mycorrhiza infection on
nitrate and ammonium uptake capacity were very similar.
Roots grown with high levels of nutrients had lower unit
nutrient uptake capacities. AM colonisation also
in-creased potential nitrate uptake rates per unit of biomass
of the yellow root parts (Fig. 2), where colonisation was
more intense (Table 2). The result was that the uptake
capacity of the yellow parts of the roots of plants grown
under low nutrient supply and infected by AM became
similar to that of the white parts of the root (Fig. 3).
Neither mycorrhiza nor the nutrient level had a significant
effect on unit inorganic nitrogen uptake by the heavily
pigmented parts of the roots (Fig. 2).
At low nutrient levels the main effect of the
mycor-rhiza was to increase the nitrogen uptake capacity of the
Table 2 Root fresh weight (g) of the three root zones consid-ered (white, yellow and brown) and the respective percentage in relation to the fresh weight of the entire root system of the plant according to treatments. Values with the same letter are not significantly different ac-cording to Fisher’s LSD test at P=0.05. Probabilities from the ANOVA are given below (df=1, 20). For abbreviations, see Fig. 1
Treatment Root Fresh weight (g) Fresh weight (% of total)
White Yellow Brown White Yellow Brown
NML 1.03 b 2.77 b 2.57 a 17 43 40 NMH 0.15 c 1.57 c 1.84 b 4 44 52 ML 2.06 a 3.23 a 2.56 a 27 41 32 MH 0.45 c 2.00 c 1.71 b 11 48 41 Source P P P AM 0.010 0.004 0.041 Nutrients 0.001 0.044 0.017 AMNutrients 0.021 0.009 0.031
Table 3 The total length, white zone, yellow zone and lightly pigmented proportion (white plus yellow zones per unit root length) in root systems of C. siliqua according to treatments. Values with the same letter are not significantly different ac-cording to Fisher’s LSD test at P=0.05. Probabilities from the ANOVA are given below (df=1, 12). For abbreviations, see Fig. 1 Treatment Length (cm) White zone (cm) Yellow zone (cm) Lightly pigmented proportion NML 270 8.0 85 0.34 a NMH 330 3.9 62 0.20 b ML 350 8.5 86 0.28 ab MH 390 3.1 78 0.20 b Source P P p P AM 0.065 0.956 0.603 0.389 Nutrients 0.136 0.058 0.344 0.011 AMNutrients 0.790 0.774 0.629 0.389
Fig. 2 Nitrate and ammonium uptake rates, on a root fresh weight (FW) basis, according to root pigmentation (white, yellow and brown) and treatment. Carob seedlings [non-mycorrhizal (grey bars) or mycorrhizal (black bars)] were grown for 3 months with low or high nutrient supply to the root medium. Values of nitrate (low and high nutrient) or ammonium (low and high nutrient) uptake rates with the same letter are not significantly different according to Fisher’s LSD test at P =0.05
younger parts of the root (white and yellow sections,
Fig. 3). Increasing the nutrient level of the root medium
lead to a reduction in the capacity of roots to take up
inorganic nitrogen, in the presence and absence of
mycorrhiza (Figs. 3, 4). Plant roots grown in low nutrient
levels and with AM colonisation were those with the
highest capacity for capturing inorganic nitrogen. These
results were confirmed by the plant content of nitrogen,
which was also higher for AM plants grown under a low
level of nutrients (Fig. 5).
The results suggested that being grown with high
nutrient levels negated the effects of AM infection, as far
as nitrogen uptake is concerned (Figs. 3, 4, 5). AM
infection improved phosphorus uptake by the plants at
low and high levels of nutrients, but this was more
noticeable at low levels of nutrients (Fig. 5). It has to be
noted, however, that the degree of colonisation by AM
fungi was lower under high levels of nutrients (Table 1),
perhaps explaining the lower phosphorus content of AM
plants grown at high nutrient levels.
AM inoculation did not significantly influence the total
root length (Table 3), the length of links, or the degree of
branching in carob root systems. There was no significant
interaction between AM inoculation and nutrients in these
variables (Table 4). The nutrient treatment did not affect
Table 4 The topology [a/E(a), where a is the number of links in the longest path between an external link and the base link, and E (a) is the expected a; and Pe/E(Pe), where Pe is the pathlength and E(Pe) the expected Pe] and link lengths [external-external (EE), external-internal (EI), internal-internal (II); mm] of C. siliqua according to treatments. Values are adjusted for the covariate log(number of tips). Values with the same letter are not signifi-cantly different according to Fisher’s LSD test at P =0.05. F -values and associated probabilities from the analysis of covariance are given below (df =1,11). For other abbreviations, see Fig. 1
Treatment a/E(a) Pe/E(Pe) EE EI II
CL 1.80 ab 1.46 1.04 1.01 0.75 a CH 1.55 ab 1.09 1.44 1.22 0.90 ab ML 2.14 a 1.79 1.31 1.18 0.80 ab MH 1.14 b 0.79 1.16 1.20 1.08 b Source P P P P P AM 0.955 0.945 0.935 0.621 0.297 Nutrients 0.029 0.053 0.612 0.484 0.047 AMNutrients 0.166 0.343 0.269 0.584 0.502 Fig. 3 Nitrate and ammonium uptake per plant, on a root pigmen-tation basis, according to the root colour (white, yellow and brown) and treatment. Carob seedlings [non-mycorrhizal (grey bars) or mycorrhizal (black bars)] were grown for 3 months with low or high nutrient supply to the root medium. Values of nitrate (low and high nutrient) or ammonium (low and high nutrient) uptake rates with the same letter are not significantly according to Fisher’s LSD test at P =0.05
Fig. 4 Nitrate and ammonium uptake rates by the root system, according to the nutrient level in the root medium. Carob seedlings [non-mycorrhizal (grey bars) or mycorrhizal (black bars)] were grown for 3 months with low or high nutrient supply to the root medium. Within panels, values with the same letter are not significantly different according to Fisher’s LSD test at P =0.05
Fig. 5 Plant nitrogen (N) and phosphorus (P) content according to the nutrient level in the root medium. Carob seedlings [non-mycorrhizal (grey bars) or [non-mycorrhizal (black bars)] were grown for 3 months with low or high nutrient supply to the root medium. Within panels, values with the same letter are not significantly different according to Fisher’s LSD test at P =0.05
the total length of root systems, but did influence the
arrangement of links within the root systems. High
nutrient supply in combination with AM inoculation
resulted in more branched (more dichotomous) root
systems with longer internal links (Table 4). Root systems
with longer internal links tended to have a greater
proportion of internal (“old”) root length compared with
absorbing length (white and yellow zones). This fits the
pattern revealed by the colour analysis (Table 3) and fresh
weight data (Table 2).
Discussion
The total biomass accumulation, per plant, is comparable
to that obtained in previous experiments (Cruz et al.
1993a, 1993b). This study revealed the improved
perfor-mance of mycorrhizal plants exposed to low levels of
nutrients in comparison with those which received
supplementary nutrients. This is in agreement with the
inhibition of AM formation in the presence of a high
concentration of nutrients, especially phosphorus (Peng et
al. 1993; Smith and Smith 1996; Graham et al. 1997).
Carob plants were not able to take advantage of high
nutrient levels in the root medium, since the growth of
plants without AM colonisation was independent of the
nutrient levels tested. Besides promotion of plant growth,
many other effects have been attributed to AM, including:
changes in the root to shoot ratio, increased leaf area,
decrease in specific leaf weight, decrease in root
longev-ity, increase or decrease in root branching depending on
plant species, and improvement in plant water relations
(Auge 2001; Majdi et al. 2001).
Control plants presented root to shoot ratios of about
1.2, a common value among seedlings of woody plants
(Martins-Lou¼o et al. 1996). The possession of a large
root system may be advantageous for the sclerophyllous,
evergreen trees and shrubs which often dominate the
vegetation of Mediterranean-type ecosystems.
Neverthe-less AM association may increase nutrient acquisition
allowing a relatively smaller biomass allocation to the
roots in order to optimise plant growth (Lambers et al.
1995).
Special attention was paid to differentiating roots
based on their pigmentation, to identify possible relations
between pigmentation and root physiological activity.
Browning (increase in root pigmentation) is concurrent
with but not caused by suberisation, a process that might
reduce the absorption efficiency of the roots (Lpez et al.
2001). Our results are is agreement with those of Comas
et al. (2000), who found that the onset of browning
generally indicated death of the roots as a “functional
organ”.
Carob plants grown with low levels of nutrients
maintained more of their root biomass in the lightly
pigmented portions. This was shown by both the colour
analysis of root length and by the fresh weight of roots
manually dissected by colour. This root architecture
would seem to be advantageous for maintaining a high
nutrient uptake capacity in conditions of low resource
availability. The greater proportion of lightly pigmented
roots was achieved, in part, by the lower internal (heavily
pigmented) link length under low nutrients, with or
without the AM association. Root systems were also more
herringbone under low nutrients. In herringbone systems
there are a slightly greater number of internal links for a
system of given magnitude (Fitter 1991). For example, a
purely herringbone system with eight external links
(magnitude=8) has seven internal links, while a purely
dichotomous system of the same magnitude has six
internal links. Thus, if all links were the same length, it
would be expected that the herringbone system would
have a greater proportion of internal, or heavily
pigment-ed, root length. However, the difference in the internal
number of links in dichotomous and herringbone systems
is small in comparison with the influence of internal link
length. This suggests that the higher proportion of lightly
pigmented root under low nutrients is related to the
shorter length of the internal links and not changes in the
topological index.
Root systems were more dichotomous under high
nutrient conditions, in agreement with the majority of the
literature, due to greater local proliferation under high
nutrients. AM association has been shown to induce
modifications in root system architecture and in root
morphogenesis (Berta et al. 1990; Schellenbaum et al.
1991; Bouma et al. 2001). The effects seem to be
dependent on fungus and plant species, as well as on
environmental conditions (Smith and Read 1997; Bouma
et al. 2001; Gao et al. 2001). Root architecture of carob
seedlings seemed to be more responsive to nutrient levels
than to AM association.
The importance of AM in nitrogen uptake and
metabolism is still controversial. It seems that the external
mycelium of AM fungi can take up organic as well as
inorganic nitrogen, and that it shows a preference for
ammonium as the inorganic nitrogen source (Whipps
2001). Much less is known about AM (fungi plus plant)
uptake rates and preferences (Smith and Read 1997).
Nitrate and ammonium uptake capacities decrease
from the apex to the more mature parts of carob roots
(Cruz et al. 1995). This gradient was still visible when
assessed on the basis of root pigmentation, with higher
uptake rates through the white and yellow parts of the
root. Roots growing in the presence of low levels of
nutrients displayed higher nitrogen uptake capacities on a
root weight basis, in agreement with the majority of
studies (Bloom et al. 1993; Crawford 1995). This is due to
an increase in the number of transporters per unit root
area in the presence of low concentrations of nutrients
(Bloom et al. 1993; George et al. 1995). The higher nitrate
uptake rates observed for the elongation and younger zone
(yellow parts of the root) of the mycorrhizal plants grown
with low levels of nutrients may be due to the presence of
the fungi, since these areas have the highest rates of AM
colonisation.
For control and AM plants grown under low nutrient
availability, the elongation zone is that which contributes
most to nitrate and ammonium uptake. It is also clear that
the presence of high levels of nutrients results in a
decrease in nitrate and ammonium uptake potential, due
to both a decrease in the nitrogen uptake capacity per root
length and a reduction of the areas that contribute most to
nutrient uptake. These calculations are in agreement with
the total nitrogen content of the plant, which was also
much more improved by AM association in the presence
of low nutrient levels. Several works have shown that
mycorrhizal fungi are involved in the uptake and transfer
of inorganic nitrogen (Smith and Read 1997). The
significance of that contribution to plant nutrition is more
controversial. Most attention has been focused on the
ability of arbuscular mycorrhizae to increase plant growth
by enhancing phosphorus uptake (Gange 2000), relieving
the plant from phosphorus stress (Fitter and Hay 2002).
AM association increased the nitrogen content of carob
grown under low nutrient levels by approximately 100%,
and biomass accumulation by 30%, showing that the
arbuscular mycorrhiza was important for the nitrogen
nutrition of the plant. Considering that the increment in
the phosphorus content of AM plants gown at high levels
of nutrients was not translated into an increment of plant
biomass accumulation or nitrogen content, the increment
in nitrogen content and biomass of AM plants grown at
low levels of nutrients cannot be exclusively attributed to
a better phosphorus status of the plants. Therefore, it
seems that nitrogen and phosphorus acquisition are
influenced by AM association in different ways.
We conclude that nutrient availability had a much
stronger effect on root architecture than the arbuscular
mycorrhiza, and that the latter increased nitrogen uptake
capacity and plant nitrogen content under low levels of
nutrients. The results suggest that in low-resource
envi-ronments, root system physiology adapts to maximise
uptake capacity, and root system morphology adapts to
maximise active uptake length. The role of the arbuscular
mycorrhiza was to increase the uptake capacity of the
active zone. AM colonisation was higher in low-resource
environments; this tendency appears to have additional
growth benefits to the carob plants unrelated to nutritional
aspects. This study is a first step in relating root
architecture and mycorrhizal colonisation to the
function-al aspects of nutrient acquisition.
Acknowledgements The Anglo-Portuguese Joint Research Pro-gramme—Treaty of Windsor Programme celebrated between the British Council and Conselho de Reitores das Universidades Portuguesas—supported this work. The Scottish Agricultural College receives financial support from the Scottish Executive Environment and Rural Affairs Department.
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